The goal of Project 2 is to study the roles of the growth factors IGF-I and insulin in th initial formation of the limb bud from the lateral plate of the chick embryo; in the induction of the AER; and, in the subsequent reciprocal interactions between the AER and subridge mesoderm that are responsible for continued outgrowth of the limb. Preliminary studies indicate that IGF-I is capable of inducing the formation of limb buds from the prospective limb-forming region of the lateral plate of the chick embryo at a stage of development when this region has not yet acquired the ability to form limbs independent of medial embryonic tissues. Thus, the role of IGF-I as well as insulin, in the inductive signalling cascade between medial tissues (intermediate mesoderm, somitic mesoderm, and Hensen's node) and the prospective limb-forming region of the lateral plate that endows presumptive limb mesoderm with limb-forming region of the lateral plate that endows presumptive limb mesoderm with limb-forming capacity, will be investigated. It will also be determined if IGF-i and insulin can induce limb formation from the normally non-limb-forming flank region of the lateral plate, and if IGF-I and insulin can induce an AER in flank ectoderm in vitro and in vivo. The role of IGF-I and insulin in the AER/subridge interactions required for continued limb outgrowth following limb formation will also be examined. Preliminary in vitro studies indicate that these growth factors are involved in promoting the outgrowth and suppressing the differentiation of the subridge mesoderm in response to the AER, and also possibly in AER activity. The role of IGF-I and insulin in the maintenance and/or regulation of AER activity will be studied, and it will be determined if these growth factors can promote the outgrowth and morphogenesis of limb mesoderm in the absence of the AER in vivo. The regulatory relationships between IGF-I, insulin, and FGFs, which have been implicated in mediating the outgrowth and patterning effects of the AER, will be studied.